Proline modulates the effect of bisphosphonate on calcium levels and adenosine triphosphate production in cell lines derived from bovine Echinococcus granulosus protoscoleces.

Bisphosphonates have been proposed as pharmacological agents against parasite and cancer cell growth. The effect of these compounds on helminthic cell viability and acellular compartment morphology, however, has not yet been studied. The effects of different types of bisphosphonates, namely etidronate (EHDP), pamidronate (APD), alendronate (ABP), ibandronate (IB) and olpadronate (OPD), and their interaction with amiloride, 1,25-dihydroxycholecalciferol (D3) and proline were evaluated on a cell line derived from bovine Echinococcus granulousus protoscoleces (EGPE) that forms cystic colonies in agarose. The EGPE cell line allowed testing the effect of bisphosphonates alone and in association with other compounds that could modulate calcium apposition/deposition, and were useful in measuring the impact of these compounds on cell growth, cystic colony formation and calcium storage. Decreased cell growth and cystic colony formation were found with EHDP, IB and OPD, and increased calcium storage with EHDP only. Calcium storage in EGPE cells appeared to be sensitive to the effect of amiloride, D3 and proline. Proline decreased calcium storage and increased colony formation. Changes in calcium storage may be associated with degenerative changes of the cysts, as shown in the in vitro colony model and linked to an adenosine triphosphate (ATP) decrease. In conclusion, bisphosphonates could be suitable tempering drugs to treat cestode infections.

Amifostine (WR2721) confers DNA protection to in vivo cisplatin-treated murine peripheral blood leukocytes.

Amifostine [S-2-3-aminopropyl amino ethyl phosphorotioic acid], a modulator agent for antineoplastic drugs involved in free radicals generation has given controversial results in cisplatin treated leukocytes in vitro. We have evaluated the amifostine protection over leukocytes in vivo, using comet assay. Groups of five OF1 male mice were given one of three doses of amifostine (56, 105 and 200 mg/Kg) after a cisplatin single injection (10 mg/Kg). Serum malonyldialdehyde levels, catalase and superoxide dismutase activity were also evaluated. Amifostine showed significant DNA protection (p< 0.01) at the two lower doses evaluated. Malonyldialdehyde decreased in all amifostine treatments with respect to cisplatin while antioxidant enzyme activities remained unchanged. However, DNA migration increased with the highest amifostine dose; in fact highest dose of amifostine did no protect damage caused by cisplatin this result have implications on amifostine treatment schedules in clinical practice.

Anti-Trypanosoma cruzi effects of cyclosporin A derivatives: possible role of a P-glycoprotein and parasite cyclophilins.

Cyclophilins are target molecules for cyclosporin A (CsA), an immunosuppressive antimicrobial drug. We have previously reported the in vitro anti-Trypanosoma cruzi activity of H-7-94 and F-7-62 non-immunosuppressive CsA analogues. In this work, we continue the study of the parasiticidal effect of H-7-94 and F-7-62 CsA analogues in vitro and in vivo and we analyse 3 new CsA derivatives: MeIle-4-CsA (NIM 811), MeVal-4-CsA (MeVal-4) and D-MeAla-3-EtVal-4-CsA, (EtVal-4). The most efficient anti-T. cruzi effect was observed with H-7-94, F-7-62 and MeVal-4 CsA analogues evidenced as inhibition of epimastigote proliferation, trypomastigote penetration, intracellular amastigote development and in vivo T. cruzi infection. This trypanocidal activity could be due to inhibition of the peptidyl prolyl cis-trans isomerase activity on the T. cruzi recombinant cyclophilins tested. Furthermore, CsA and F-7-62 derivative inhibited the efflux of rhodamine 123 from T. cruzi epimastigotes, suggesting an interference with a P-glycoprotein activity. Moreover, H-7-94 and F-7-62 CsA analogues were not toxic as shown by cell viability and by aminopyrine-N-demethylase activity on mammalian cells. Our results show that H-7-94, F-7-62 and MeVal-4 CsA analogues expressed the highest inhibiting effects on T. cruzi, being promissory parasiticidal drugs worthy of further studies.

Ionic fluxes through myelin membrane vesicles.

Technical difficulties and a lack of reproducibility in procedures aimed at the production of myelin vesicles have delayed functional studies on membrane transport through myelin. Myelin vesicles could provide an excellent model for the study of the transport of ions and water, etc., across this type of membrane. They could also help improve our understanding of the molecular functions of the myelin sheath. In this investigation, a novel, nonaggressive method of producing central nervous system myelin vesicles is presented. Purified bovine myelin was incubated with iminodiacetic acid (an insoluble chelating agent that is easy to remove and does not interfere with further functional assays), and rendered insoluble on 1% crosslinked polystyrene beads (Chelex-100). Myelin vesicles obtained were impermeable to sugars (sucrose, glucose, and galactose), but showed a degree of permeability towards potassium salts as determined by light-scattering. Further experiments with fluorescent probes revealed an electrogenic K+ influx, as measured by oxonol V fluorescence quenching, and a significant H+ permeability measured using the pH-sensitive probe acridine orange. H+ permeability was not detected in control liposomes made from the same endogenous myelin lipids without protein. The results are discussed with reference to previous studies performed using purified myelin proteins in reconstituted systems. The relevance of these results with respect to ionic transport across myelin membrane is discussed.

Relationship between the content of [14C]glucose-derived monosaccharides in glycoprotein oligosaccharide chains and the state of enterocytic differentiation of HT-29 cells.

The HT-29 cell line derived from a human colon adenocarcinoma has a glucose-dependent state of differentiation which is negatively correlated with the presence of D-glucose in the culture medium. The contribution of glucose to the biosynthesis of N-glycan chains, as a function of the differentiation state of HT-29 cells, was shown by: (a) [14C]glucose incorporation by undifferentiated HT-29 cells being lower after 2 h and higher after 19 h of metabolic labeling than that by differentiated cells; (b) a lack of glucose in the culture medium of undifferentiated HT-29 cells diminishing [14C]glucose incorporation into glycan chains, but not changing the glucose distribution between lipid- and protein-linked saccharides; (c) glucose behavior in undifferentiated HT-29 cells being not related to mannose-glycan metabolism, as the high-mannose compounds labeled with glucose and observed by HPLC showing a different distribution associated with the duration of glucose labeling; and (d) glucose being interconverted into other monosaccharide-glycan constituents in proportions different in differentiated and undifferentiated cell populations.

Localization of tissue copper in mouse mammary tumors.

The purpose of this study was to determine the copper deposition and localization during the evolution of two murine mammary adenocarcinomas. In the normal tissue, the copper was located within the cytoplasm, whereas it was intra- and perinuclear in the tumors. The more angiogenic and metastatic tumor showed the higher percentage of copper-positive cells. In the tumor, copper deposits correlated well with its angiogenic and metastatic ability, but additional factors would be required for the process to be induced.

Copper histochemistry of 5 murine tumors and their respective metastases.

Tissue copper content has been evaluated in 4 murine mammary adenocarcinomas, 1 murine lung adenocarcinoma and their respective metastases. Histochemical techniques have been used to analyze copper distribution in tumor tissues. It is observed that the degree of copper staining is inversely related to tumor differentiation. As the copper level reflects one of the metabolic changes in the host carrying the tumor, it is suggested that it could be used as a good marker for tumor differentiation.

Serum and tissue copper content in two mammary adenocarcinomas with different biological behaviour.

We have determined serum copper levels in BALB/c female mice subcutaneously inoculated with two related mammary adenocarcinoma tumors. Serum copper content increased progressively with tumor size in animals bearing either variant, and reached levels up to four times those of control mice. In contrast, copper levels detected by cytochemistry in tumor cells are higher in the slow growing tumor variant. It is suggested that the stronger angiogenic effect previously described for this variant could be related to its higher cellular copper content.